The present invention relates to an isolated dna molecule encoding a cytochrome p450lpr polypeptide as well as the isolated cytochrome p450lpr polypeptide itself. The dna molecule can be inserted as a heterologous dna in an expression vector forming a recombinant dna expression system for producing the polypeptide. Likewise, the heterologous dna, usually inserted in an expression vector to form a recombinant dna expression system, can be incorporated in a host cell to achieve this objective.
The dna molecule of the present invention can be utilized for control of larval or adult insects, bioremediation of insecticides, and conferring pesticide resistance to crop plants.
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1. An isolated dna molecule encoding cytochrome p450lpr polypeptide, wherein said dna molecule is either the nucleotide sequence of SEQ. ID. NO. 1 or dna variants of said sequence which retain cytochrome p450lpr dependent enzymatic activity.
7. A cell transformed with a heterologous dna molecule encoding cytochrome p450lpr, wherein said heterologous dna molecule is either the nucleotide sequence of SEQ. ID. NO. 1 or dna variants of said sequence which retain cytochrome p450lpr dependent enzymatic activity.
2. An isolated dna molecule according to
3. An expression system comprising the isolated dna molecule of
4. An expression system according to
5. An expression system according to
6. An expression system according to
8. A cell according to
9. A cell according to
10. A cell according to
11. A method of adult insect control comprising: treating adult insects with a vector comprising the dna molecule of
12. A method according to
13. A method according to
14. A method according to
15. A method according to
17. A transgenic plant according to
18. A transgenic plant according to
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The subject matter of this application was made with support from the United States Government (National Institutes of Health Grant No. R01 GM47835-01 and United States Department of Agriculture Grant No. 9001168).
This is a division of application Ser. No. 08/241,388 filed on May 10, 1994 .
The present invention relates to the cytochrome P450lpr gene and its uses.
The microsomal cytochrome P450-dependent monooxygenases (hereafter called "P450 monooxygenases") are an extremely important metabolic system involved in the detoxication of xenobiotics such as drugs, pesticides, and plant toxins; and in the regulation of endogenous compounds such as hormones, fatty acids, and steroids. P450 monooxygenases are found in almost all aerobic organisms, including organisms as diverse as plants, insects, mammals, birds, and fungi. See U.S. Pat. Nos. 4,766,068 to Oeda, et al., 5,164,313 to Gelboin, et al., and 5,212,296 to Dean, et al. In eucaryotes, P450 monooxygenases are typically found in the endoplasmic reticulum of metabolically active tissues. The two most important components of the P450 monooxygenases are cytochrome P450, which acts as the substrate binding protein (and terminal oxidase), and NADPH-cytochrome P450 reductase (P450 reductase), which transfers electrons from NADPH to cytochrome P450. Cytochrome b5 may have a role in P450 monooxygenase activity by donating an (i.e., the second) electron to P450 (via cytochrome b5 reductase) or by modulating P450 monooxygenase activity through inhibitory or stimulatory interactions with P450. See S. Kawano, et al., J. Biochem., 102:493 (1987) and I. Golly, et al., Arch. Biochem. Biophys., 260:232 (1988), which are hereby incorporated by reference. However, the exact importance of cytochrome b5 is still questioned, because it is not required for activity in most reconstituted P450 monooxygenase systems. The role of cytochrome b5 and Cytochrome b5 reductase in P450 monooxygenase activity in insects is poorly understood.
P450 monooxygenases are capable of oxidizing a bewildering array of xenobiotics, E. Hodgson, "Microsomal mono-oxygenases," Comprehensive Insect Physiology Biochemistry and Pharmacology, Vol. 11, G. A. Kerkut and L. I. Gilbert, eds., Pergamon Press, Oxford (1985), which is hereby incorporated by reference. This remarkable breadth of utilizable substrates is due to the large number of cytochrome P450 forms that are expressed in each organism. For instance, over 13 P450s have been isolated from rats, F. P. Guengerich, "Enzymology of Rat Liver Cytochrome P450, in: "Mammalian Cytochrome P450", Vol. 1, F. P. Guengerich, ed., CRC Press, Boca Raton, Fla. (1987), which is hereby incorporated by reference, and 221 cytochrome P450 cDNA sequences have been described mostly from mammalian systems, D. R. Nelson, et al., "The P450 Superfamily: Update on New Sequences, Gene Mapping, Accession Numbers, Early Trivial Names of Enzymes, and Nomenclature," DNA Cell Biol., 12:1-51 (1993), which is hereby incorporated by reference. The specificity of the P450 monooxygenase system, therefore, is dependent on the P450 cytochrome(s) present, many with apparently overlapping specificity. This complexity requires that individual cytochrome P450 forms be isolated in order to understand and characterize their contribution to important metabolic functions.
The P450 monooxygenases in insects are extremely important for growth, development, feeding, resistance to pesticides, and tolerance to plant toxins, M. Agosin, "Role of Microsomal Oxidation in Insecticide Degradation", Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 12, G. A. Kerkut and L. I. Gilbert, eds., Pergamon Press, Oxford (1985) and E. Hodgson, "The Significance of Cytochrome P450 in Insects," Insect Biochem., 13:237-246 (1983), which are hereby incorporated by reference. Furthermore, P450 monooxygenases are intimately involved in the synthesis and degradation of insect hormones and pheromones including 20-hydorxyecdysone and juvenile hormone, M. Agosin, "Role of Microsomal Oxidation in Insecticide Degradation," Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 12, G. A. Kerkut and L. I. Gilbert, eds., Pergamon Press, Oxford (1985), which is hereby incorporated by reference. Insect P450 monooxygenases can be detected in a wide range of tissues. Highest P450 monooxygenase activities are usually associated with the midgut, fat body, and Malpighian tubules, Id. and E. Hodgson, "The Significance of Cytochrome P450 in insects, Insect Biochem., 13:237-246 (1983), which are hereby incorporated by reference. Dramatic variations in the levels of cytochrome P450 and monooxygenase activity are seen during the development of most insects, Id. and D. R. Vincent, et al., "Cytochrome P450 in Insects. 6. Age Dependency and Phenobarbital Inducibility of Cytochrome P-450 Reductase and Monooxygenase Activity in Susceptible and Resistant Strains of Musca domestica," Pestic. Biochem. Physiol., 23:171-81 (1985), which are hereby incorporated by reference. In general, P450 levels are undetectable in eggs, rise and fall in each larval instar, are undetectable in pupae, and are expressed at high levels in adults. M. Agosin, "Role of Microsomal Oxidation in Insecticide Degradation," Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 12, G. A. Kerkut and L. I. Gilbert, eds., Pergamon Press, Oxford (1985), which is hereby incorporated by reference.
The role of monooxygenases in insecticide resistance first became apparent in the early 1960s, when Eldefrawi, et al., "Methylenedioxyphenyl Derivatives as Synergists for Carbamate Insecticides in Susceptible, DDT-, and Parathion-Resistant House Flies," J. Econ. Entomol., 53:231-34 (1960), which is hereby incorporated by reference, showed that resistance to carbaryl could be abolished by the methylenedioxphenyl cytochrome P450 inhibitor sesamex. Additional evidence of monooxygenase-based resistance quickly accumulated. G. P. Georghiou, et al., "The Absorption and Metabolism of 3-isopropyl-phenyl N-methylcarbamate by Susceptible and Carbamate-Selected Strains of House Flies," J. Econ. Entomol., 54:231-33 (1961), G. P. Georghiou, et al., "The Development and Characterization of Resistance to Carbamate Insecticides in the House Fly, Musca domestica," J. Econ. Entomol., 54:132-140 (1961), and R. D. Schonbrod, et al., "Hydroxylation as a Factor in Resistance in House Flies and Blow Flies," J. Econ. Entomol., 58:74-78 (1965), which are hereby incorporated by reference. In 1967, Tsukamoto and Casida (M. Tsukamoto, et al., "Metabolism of Methylcarbamate Insecticides by the NADPH2 -requiring Enzyme System from Houseflies," Nature London, 213:49-51 (1967), which is hereby incorporated by reference) showed that carbamate-resistant house flies exhibited increased ability, compared to susceptible flies, to perform oxidative hydroxylations, N-demethylations, O-demethylations, epoxidations, and desulfurations. Soon after, other workers showed the diazinon-resistant Fc strain had increased ability to oxidize DDT, F. J. Oppenoorth, et al., "DDT Resistance in the Housefly Caused by Microsomal Degradation", Ent. Exp. Appl., 11:81-93 (1968), which is hereby incorporated by reference, and aldrin and naphthalene, R. D. Schonbrod, et al., "Microsomal Oxidases in the House Fly: A Survey of Fourteen Strains," Life Sci. 7:681-688 (1968), which is hereby incorporated by reference. Increased ability to metabolize many substrates was shown in other insecticide-resistant house fly strains such as Rutgers diazinon-resistant strain, M. D. Folsom, et al., "Biochemical Characteristics of Microsomal Preparations from Diazinon-resistant and -susceptible Houseflies," Life Sci. 9:869-875 (1970), which is hereby incorporated by reference. In addition, increased levels of total cytochrome P450 were found in several insecticide-resistant strains such as Diazinon-R, Fc, R-Baygon, Dimethoate-R, Orlando-R, Malathion-R, and Ronnel-R. E. Hodgson, "Microsomal Mono-oxygenases," Comprehensive Insect Physiology Biochemistry and Pharmacology, G. A. Kerkut, et al., eds., Pergamon Press, Oxford, 11:225-321 (1985), which is hereby incorporated by reference.
We now know that insects commonly become resistant to insecticides due to increased detoxication mediated by the cytochrome P450 monooxygenase system. This resistance mechanism is very important, because it can confer both high levels of resistance, L. B. Brattsten, et al., "Insecticide Resistance: Challenge to Pest Management and Basic Research," Science, 231:1255-60 (1986) and J. G. Scott, et al., "Mechanisms Responsible for High Levels of Permethrin Resistance in the House Fly," Pestic. Sci., 17:195-206 (1986), which are hereby incorporated by reference, and may also confer cross-resistance to unrelated compounds due to the breadth of substrates the P450 monooxygenases can metabolize. J. G. Scott, "Insecticide Resistance in Insects," Handbook of Pest Management, Vol. 2, D. Pimentel, ed., CRC Press, Boca Raton, Fla. (1991), which is hereby incorporated by reference. Furthermore, P450 monooxygenase-mediated detoxication has been found as a mechanism of resistance in a large number of important pests. C. F. Wilkinson, "Role of Mixed-function Oxidases in Insecticide Resistance," Pest Resistance in Pesticides, G. P. Georghiou and T. Saito, eds., Plenum Press, New York (1983), which is hereby incorporated by reference.
Purification of a P450 from insects in useful quantity and quality remained elusive for many years due to the difficulties encountered in insect cytochrome P450 purification. E. Hodgson, "Microsomal mono-oxygenases," Comprehensive Insect Physiology Biochemistry and Pharmacology, Vol. 11, G. A. Kerkut and L. I. Gilbert, eds., Pergamon Press, Oxford (1985) and C. W. Fisher, et al., "Partial Purification and Characterization of Phenobarbital-Induced House Fly P-450," Arch. Insect Biochem. Physiol., 1:127-38 (1984), which are hereby incorporated by reference. R. D. Schonbrod, et al., "The Solubilization and Separation of Two Forms of Microsomal cytochrome P-450 from the House Fly, Musca domestica," Biochem. Biophys. Res. Comm., 64:829-833 (1975), which is hereby incorporated by reference, reported resolution of two forms of low specific content (impure or damaged cytochrome P450 preparations) but produced early evidence of multiplicity of cytochrome P450 in house flies. An early attempt, J. Capdevila, et al., "Multiple Forms of Housefly Cytochrome P-450," Microsomes and Drug Oxidations, V. Ulrich, ed., Pergamon Press, New York (1977), which is hereby incorporated by reference, using uninduced, susceptible house flies was remarkably successful, isolating one P450 with high specific content (13.9 nmol cytochrome P450/mg protein) but impure based on gel electrophoresis. This method required seven open-column chromatographic steps and apparently was not pursued further. The resolution of several impure cytochrome P450 preparations was reported by S. J. Yu, et al., "Cytochrome P-450 in insects. 1. Differences in the Forms Present in Insecticide Resistant and Susceptible House Flies," Pestic. Biochem. Physiol., 12:239-48 (1979), which is hereby incorporated by reference. A. F. Moldenke, et al., "Cytochrome P-450 in Insects 4. Reconstitution of Cytochrome P-450-dependent monooxygenase Activity in the House Fly," Pestic. Biochem. Physiol., 21:358-67 (1984), which is hereby incorporated by reference, resolved two crude cytochrome P450 fractions and reconstituted the Oxidase activity with purified cytochrome P450 reductase to show that different cytochrome P450 fractions have different metabolic capabilities. In the same year, C. W. Fisher, et al., "Partial Purification and Characterization of Phenobarbital-. Induced House Fly P-450," Arch. Insect Biochem. Physiol., 1:127-38 (1984), which is hereby incorporated by reference, reported a partially pure preparation from the Rutgers diazinon-resistant strain with a specific content of 10 nmol/mg and partially characterized it. M. J. J. Ronis, et al., "Characterization of Multiple Forms of Cytochrome P-450 From an Insecticide Resistant Strain of House Fly (Musca domestica)," Pestic. Biochem. Physiol., 32:74-90 (1988), which is hereby incorporated by reference, partially purified several P450s from the Rutgers strain with specific contents ranging between 2.5 and 7 nmol/mg and showed that they could, with limited success, be reconstituted with mammalian cytochrome P450 reductase. However, a biochemically useful purification of a cytochrome P450 from an insect, and more importantly from an insecticide-resistant house fly, remained elusive for many years.
In 1989, the purification of a major cytochrome P450, termed P450lpr, from LPR house flies to apparent electrophoretic homogeneity was reported in G. D. Wheelock, et al., "Simultaneous Purification of a Cytochrome P-450 and Cytochrome b5 from the House Fly Musca domestica L.", Insect Biochem., 19:481-489 (1989), which is hereby incorporated by reference. This P450 runs as a single band at 54.3 kDa by SDS-PAGE, corresponding to a major band in LPR, and a phenobarbital-inducible band in wild-type (susceptible) flies. It has a carboxy ferrocytochrome absorbance maximum at 447 nm with no apparent peak at 420 (i.e., no denatured P450), has a high specific content (14.4 nmol/mg protein), and can be readily isolated in substantial quantities. Id. The N-terminal sequence of 15 amino acids for cytochrome P450lpr is disclosed in J. G. Scott, et al., "Characterization of a Cytochrome P450 Responsible for Pyrethroid Resistance in the Housefly," Molecular Basis of Insecticide Resistance: Diversity Among Insects, Symposium Series 505, C. J. Mullin and J. G. Scott, eds., American Chemical Society, Washington, D.C. (1992), which is hereby incorporated by reference. This sequence shares no homology with published P450 sequences. P450lpr appears to be a single cytochrome P450, because it cannot be resolved into multiple components chromatographically, immunologically, or electrophoretically. G. D. Wheelock, et al., "Immunological Detection of Cytochrome P450 from Insecticide Resistant and Susceptible House Flies (Musca domestica)," Pestic. Biochem. Physiol., 38:130-39 (1990), which is hereby incorporated by reference. A polyclonal antiserum was raised in rabbits using purified cytochrome P450lpr protein as the antigen and was shown to be monospecific for cytochrome P450lpr. This antiserum has proven extremely useful in the characterization of P450lpr.
Immunological studies of insect P450s have shown limited homology with P450s from other classes. G. D. Wheelock, et al., "Expression of Cytochrome P-450lpr is Developmentally Regulated and Limited to House Fly," J. Biochem. Toxicol., 6:239-246 (1991), which is hereby incorporated by reference, surveyed several animals for the presence of P450lpr. Adult face flies, stable flies, and Drosophila all gave negative immuno-staining responses, as did larval Drosophila. Representatives of Hymenoptera (the honey bee and carpenter ant), Lepidoptera (the cabbage looper and tobacco hornworm), Orthoptera (the German cockroach), and Acari (the two spotted spider mite) did not immuno-stain. Phenobarbital induction of P450 was obtained in the face fly, stable fly, Drosophila adults, tobacco hornwork, German cockroach and honey bee. This induction did not produce immuno-stainable P450. P450 monooxygenase-mediated, insecticide-resistant arthropod strains tested include Hikone-R Drosophila, Dursban-R German cockroaches, and two spotted spider mites. Insecticide resistance did not confer expression of immunologically recognized cytochrome P450 in these arthropods. Id. Microsomes from rat or mouse liver were tested with anti-P-450lpr for cross-reactivity. No immuno-staining bands in blots from corn oil-treated, 3-methylchol-anthrene-treated or phenybarbital-treated rat liver microsomes were found. Additionally, no reaction was seen with corn oil/treated or benzo(e)pyrene-treated mouse liver microsomes. Id. It was concluded that P-450lpr is likely restricted to house flies due to the total lack of cross-reactivity to anti-P450lpr in any of the wide range of species tested.
Cytochrome P450lpr is expressed in both male and female adult LPR house flies. Id. Microsomes from males had a specific content of 0.53 nmol P450lpr /total nmol of cytochrome P450 or 0.37 nmol P450lpr /mg protein. Microsomes from female flies had 0.53 nmol P450lpr /nmol P450 or 0.14 nmol P450lpr /mg protein. Thus, P450lpr represented the same fraction of total P450 in both female and male microsomes but less on a per mg protein basis in females. Id. This agrees with previous reports examining the specific content of total cytochrome P450 (or cytochrome P450-dependent enzymatic activity) where only minor differences between adult male and female house flies have been found. M. Agosin, "Role of Microsomal Oxidation in Insecticide Degradation," Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 12, G. A. Kerkut and L. I. Gilbert, eds., Pergamon Press, Oxford (1985), which is hereby incorporated by reference.
It appears that cytochrome P450lpr is developmentally regulated in the LPR strain. G. D. Wheelock, et al., "Expression of Cytochrome P-450lpr is Developmentally Regulated and Limited to House Fly," J. Biochem. Toxicol., 6:239-246 (1991), which is hereby incorporated by reference. P450lpr was present in adults of all ages, from 0-3 hr to 5-6 days post emergence as detected by SDS-PAGE immuno-blotting. In contrast, microsomes from 1, 2, 3, 4, 5, and 6 day old LPR larvae revealed no immuno-staining material corresponding to P450lpr. Id. P450lpr was expressed at barely detectable levels early in pupal development (i.e., between 24-48 hrs. after pupation), and was present at low levels in all pulal stages thereafter. M. E. McManus, et al., "Identification and Quantitation in Human Liver of Cytochrome P-450 Analogous to Rabbit Cytochrome P-450 Forms 4 and 6," Xenobiotica, 18:207-16 (1988), which is hereby incorporated by reference. Therefore, it appears that P450lpr is first synthesized in pupae, with significant P450lpr expression limited to adults, but it is not otherwise sex or age specific.
P450 isoforms are found in most tissues throughout the house fly abdomen. The relative abundance of P450lpr in abdominal tissues from adult female house flies is fat body proximal intestine>reproductive system. S. S. T. Lee, et al., "Tissue Distribution of Microsomal Cytochrome P450 Monooxygenases and Their Inducibility by Phenobarbital in the House Fly, Musca domestica L.," Insect Biochem. Molec. Biol., 22:699-711 (1992), which is hereby incorporated by reference. Interestingly, P450lpr was found not only in tissues that have relatively high environmental exposure but also in the female reproductive system. This suggests that P450lpr may not be limited to xenobiotic detoxication but may also be important for other physiological functions.
The average amount of P450lpr in LPR microsomes, as a percentage of the total P450 was determined to be 68% by quantitative immuno-electrophoresis, G. D. Wheelock, et al., "Immunological Detection of Cytochrome P450 from Insecticide Resistant and Susceptible House Flies (Musca domestica)," Pestic. Biochem. Physiol., 38:130-39 (1990), which is hereby incorporated by reference, suggesting that P450lpr was the major cytochrome P450 in microsomes from the pyrethroid-resistant LPR strain of the house fly. In microsomes from the insecticide-susceptible S+ strain, P450lpr was found to be a minority of the total cytochrome P450, comprising only 6.5% of the total. Id. A calculation from the specific contents results in an estimation of 44-fold higher levels of immunologically reactive cytochrome P450 in LPR microsomes compared to S+ microsomes. G. D. Wheelock, et al., "Expression of Cytochrome P-450lpr is Developmentally Regulated and Limited to House Fly," J. Biochem. Toxicol., 6:239-246 (1991), which is hereby incorporated by reference. It appears that the immunoreactive proteins in the S+ and LPR strains are identical, because they are indistinguishable by electrophoretic, chromatographic, or immunological techniques and have the same N-terminal amino acid sequence. G. D. Wheelock, et al., "Immunological Detection of Cytochrome P450 from Insecticide Resistant and Susceptible House Flies (Musca domestica)," Pestic. Biochem. Physiol., 38:130-39 (1990), Which is hereby incorporated by reference.
Microsomes from insecticide-resistant and susceptible house fly strains were evaluated for the presence of P450lpr, and single immunoreactive bands were found in the resistant strains (i.e. LPR, Dairy, Kashiwagura, 3rd-Y, EPR, ASPRm, ASPRf), while the susceptible strains (i.e. aabys and S+) showed only weak reactions. Id. Additionally, cytochrome P450s were isolated and fractionated from the LPR, aabys, S+, Dairy, Kashiwagura, 3rd-Y, EPR, ASPRm, and ASPRf house fly strains using hydrophobic interaction and ion exchange HPLC. The fractionated P450s from the ion exchange step were assayed for immunoreactivity by quantitative immuno-electrophoresis. The results revealed a major cytochrome P450 peak of similar retention time associated with immunoreactivity for each strain. The immunoreactive fractions from the HPLC experiment all fused completely with P450lpr, indicating immunological identity with P450lpr. Id.
In addition to those references listed above, the following journal articles, all of which are hereby incorporated by reference, discuss studies of P450lpr : G. D. Wheelock, et al., "Anti-P450lpr Antiserum Inhibits Specific Monooxygenase Activities in LPR House Fly Microsome," The Journal of Experimental Zoology, 264:153-58 (1992); G. D. Wheelock, et al., "The Role of Cytochrome P450lpr in Deltamethrin Metabolism by Pyrethroid-Resistant and Susceptible Strains of House Flies," Pestic. Biochem. & Physiol., 43:67-77 (1992).; S. S. T. Lee, et al., "In Vitro Induction of Microsomal Cytochrome P-450 Monooxygenases by Phenobarbital in Fat Bodies of Adult House Fly, Musca domestica L.," Insect Biochem. Molec. Biol., 22 (7): 691-98 (1992); R. Hatano, et al., "Anti-P450lpr Antiserum Inhibits the Activation of Chlorpyrifos to Chlorpyrifos Oxan in House Fly Microsomes," Pestic. Biochem. & Physiol., 45:228-33 (1993); J. G. Scott, "The Cytochrome P450 Microsomal Monooxygenases of Insects: Recent Advances," Rev. Pestic. Toxicol., Vol. 2, R. M. Roe et. al., editors (1993); and J. G. Scott, et al., "Purification and Characterization of a Cytochrome P-450 From Insecticide Susceptible and Resistant Strains at Housefly Musca domestica L., Before and After Phenobarbital Exposure," Arch. Insect Biochem. & Physiol., 24:1-19 (1993).
The present invention relates to an isolated DNA molecule encoding a cytochrome P450lpr polypeptide as well as an isolated cytochrome P450lpr polypeptide itself. The DNA molecule can be inserted as a heterologous DNA in an expression vector forming a recombinant DNA expression system for producing the polypeptide. Likewise, the heterologous DNA, usually inserted in an expression vector to form a recombinant DNA expression system, can be incorporated in a host cell for expression of cytochrome P450lpr polypeptide.
The DNA molecule of the present invention can be utilized in a variety of ways. According to one use, insect larvae can be treated with a vector containing the DNA molecule of the present invention to achieve insect control. Alternatively, adult insects can be controlled by treating them with a vector comprising the DNA molecule of the present invention together with an insecticide. The DNA molecule of the present invention can also be used in a bioremediation process by applying a vector comprising that DNA molecule to an insecticide. Yet another aspect of the present invention involves transforming crop plants with the DNA molecule encoding cytochrome P450lpr polypeptide to reduce their sensitivity to pesticides.
FIG. 1 shows an in vitro cloning strategy by PCR. cDNA parts amplified from center region, A; downstream, B; upstream, C; a cDNA region examined for intron insertions in the corresponding genomic DNA, D. The arrow heads presented on A-C lines shows primers used for amplification of each cDNA part; the arrow heads under A, B, D shows the primers used for primer walking or for differential size screening of intron-involving PCR products from genomic DNA; triangles present intron insertion sites in the corresponding structure gene sequence. White and black bars present cDNA coding region and untranslated regions, respectively. The sequences of named primers are described infra in Table 1.
FIG. 2 shows the cDNA (SEQ. ID. No. 1) and deduced protein (SEQ. ID. No. 2) sequences of P450lpr. The bases are numbered from the translation initiation site as +1. Putative polyadenylation signals and the amino acid sequences primarily determined from P450lpr polypeptides are underlined. EcoRI site and the invariant or highly conserved residues in all the P450 proteins are doubly underlined. The intron insertion sites in the corresponding genomic DNA are shown with tentative numbers on the cDNA sequence.
FIG. 3 shows the genomic Southern hybridization of LPR DNA fragments. Autoradiography A, B, and C show an EcoRI DNA blot hybridized to each of the 3 cDNA probes, upstream and downstream from, and encompassing, a unique EcoRI site in the P450lpr cDNA sequence, respectively. The arrangement of the cDNA probes are shown in FIG. 1. EcoRI fragments of 2.2 and 3.5 kbp were oriented in this order.
FIG. 4 shows the alignment of housefly P450lpr with P450VI family insect members and a P450III family member. A colon denotes a residue shared among all the sequences in alignment; a dot denotes a residue shared among four sequences. Numbering on the alignment shows lpr residue positions. Numbering in parenthesis shows alignment frame positions.
FIG. 5 depicts a comparison of the amino sequence for cytochrome P450lpr and the corresponding amino acid sequence for susceptible house flies.
FIGS. 6A-6D depict a comparison of the nucleotide sequence for cytochrome P450lpr and the corresponding nucleotide sequence for susceptible house flies.
The present invention relates to an isolated DNA molecule encoding for cytochrome P450lpr polypeptide. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 as follows: ##STR1##
The DNA molecule, corresponding to SEQ. ID. No. 1, encodes for the deduced cytochrome P450lpr polypeptide or protein which corresponds to SEQ. ID. No. 2 as follows: ##STR2##
In the amino acid sequence designated as SEQ. ID. No. 2, the underlined sequences correspond to peptides known from sequencing digested protein. These sequences are separately denominated as SEQ. ID. Nos. 3-7 as follows: ##STR3##
The protein or polypeptide of the present invention is preferably produced in purified form by conventional techniques. Typically, the protein or polypeptide of the present invention is secreted into the growth medium of recombinant E. coli. To isolate the protein, the E. coli host cell carrying a recombinant plasmid is propagated, homogenized, and the homogenate is centrifuged to remove bacterial debris. The supernantant is then subjected to sequential ammonium sulfate precipitation. The fraction containing the protein of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC.
The DNA molecule encoding the cytochrome P450lpr polypeptide can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture.
Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see F. W. Studier et. al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology vol. 185 (1990), which is hereby incorporated by reference), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982), which is hereby incorporated by reference.
A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation).
Transcription of DNA is dependent upon the presence of a promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promotors differ from those of procaryotic promotors. Furthermore, eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promotors are not recognized and do not function in eucaryotic cells.
Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno ("SD") sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3'-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference.
Promotors vary in their "strength" (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promotors in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promotors may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promotors such as the T7 phage promoter, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the PR and PL promotors of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.
Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promotor, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (SD) sequence about 7-9 bases 5' to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
Once the isolated DNA molecule encoding cytochrome P450lpr polypeptide has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, and the like.
Cytochrome P450lpr is an enzyme capable of metabolizing insecticides. P450lpr detoxifies pyrethroids and activates organophosphate insecticides. These are the two most widely used classes of insecticides worldwide. Possible applications of the cytochrome P450lpr DNA molecule and polypeptide sequences of the present invention include: 1) the expression of P450lpr for use as an insecticide; 2) expression of P450lpr in an appropriate vector for use as a synergist for organophosphate insecticides; 3) expression of P450lpr in an appropriate vector as a bioremediation agent for the clean up of pyrethroid insecticide residues; and 4) engineering the P450lpr gene into plants to reduce phytotoxicity of insecticides.
One aspect of the present invention is directed to a method of insect control where insect larvae are treated with a vector containing the DNA molecule of the present invention. It has been found that cytochrome P450lpr is a developmentally regulated protein that is expressed in adults, but not in larval houseflies. This suggests that the P450lpr protein is strictly controlled (i.e., not expressed) in larvae in order to allow them to develop normally. Thus, cloning the P450lpr DNA molecule into a vector, infecting larvae with that vector, and expressing the P450lpr protein in infected larvae would be useful as an insecticide. More particularly, such expression of the DNA molecule encoding P450lpr protein would either kill larvae or radically disrupt their development. Additionally, because the protein is house fly specific, it may be lethal to other insects if expressed.
A particularly suitable vector for infecting insect larvae is the insect-specific baculovirus. When using the baculovirus system to infect larvae, it is first necessary to clone the DNA molecule coding for cytochrome P450lpr polypeptide into a baculovirus transfer vector. The baculovirus transfer vector and Autographa californica nuclear polyhedrosis virus genomic DNA are then used to coinfect host insect cells. Suitable insect cells for such transfection are Sf-9 or Sf-21 insect cells. A recombinant baculovirus can then be recovered and used to infect insect larvae in accordance with the above-described method.
Vectors containing the DNA molecule encoding for cytochrome P450lpr polypeptide can be applied directly or indirectly to insect larvae as a spray. For example, the polypeptide can be used to treat plants, soil or other habitat. It may also be used in conjunction with an attractant (e.g., a pheromone), particularly where the surface of application is not a food source to lure adult female insects to that surface where they deposit their eggs. The vector containing DNA encoding for cytochrome P450lpr polypeptide then infects either the eggs or the resulting larvae so that that DNA molecule is incorporated in the eggs or larvae. Subsequent expression of cytochrome P450lpr acts against propagation of the eggs or larvae.
Additionally, as discussed more fully infra, a gene encoding for the polypeptide can be incorporated in a plant, which would normally serve as an insect food source. In this embodiment, the expressed polypeptide would serve as an insecticide to insects consuming the plant.
In another aspect of the present invention, a vector containing the DNA molecule encoding for cytochrome P450lpr polypeptide is used to treat adult insects as part of an insect control procedure. In this embodiment of the present invention, the vector is used together with an organophosphate insecticide, because it has been found that cytochrome P450lpr polypeptide is effective in achieving activation of such insecticides. See R. Hatano, et al., "Anti-P450lpr Antiserum Inhibits the Activation of Chlorphyrifos to Chlorphyrifos Oxon in Housefly Microsomes", Pestic. Biochem. and Physiol., 45:228-33 (1993), which is hereby incorporated by reference. As a result, insects which express high levels of P450lpr are more likely to be sensitive to organophosphate insecticides. This would allow lower concentrations of the organophosphate insecticides to be used and would be useful as a strategy for control of organophosphate resistance in insects.
Suitable organophosphate insecticides for use in conjunction with the present invention include (but are not limited): parathion, malathion, azinphosmethyl, diazinon, chlorphyrifos, terbufos, and fenitrothion. Generally, organophosphate insecticides are used at levels of 0.2 to 60 pounds per acre. The vector containing the DNA molecule of the present invention can be utilized in conjunction with an organophosphate insecticide in the form of a spray, powder, or with an attractant. Again, this regime for controlling adult insects is particularly adaptable to use with the baculovirus vector, as described above.
In yet another aspect of the present invention, a vector containing the DNA molecule encoding for cytochrome P450lpr polypeptide can be applied to insecticide spills as part of a method of bioremediation. Microorganisms can be extremely useful as agents for clean-up of environmental problems, including pesticides spills. Development of suitable microorganisms involves either selecting microorganisms with a bioremediation trade or by introducing a gene into microbes to engender them with that ability. By introducing the DNA molecule encoding for cytochrome P450lpr polypeptide into an appropriate vector, it is possible to achieve bioremediation of insecticide residues. Suitable vectors are non-pathogenic bacteria.
In yet another aspect of the present invention, the DNA molecule encoding cytochrome P450lpr can be used to transform crop plants in order to reduce their sensitivity to pesticides. If crops are sensitive to certain pesticides, the types of materials used to control insects in conjunction with such crops may be severly limited. There has been a great deal of recent activity in the agrochemical industry to develop genes that confer tolerance to pesticides. As a result, compounds that might not normally be used in conjunction with a given crop due to phytotoxicity problems can be useful. Similarly, since cytochrome P450lpr is known to be useful in breaking down pesticides, expression of a gene encoding for that protein within plants will confer protection against pesticide phytotoxicity.
The isolated DNA molecule of the present invention can be utilized to confer protection against pesticide phytotoxicity for a wide variety of plants, including gymnosperms, monocots, and dicots. Although the gene can be inserted into any plant falling within these broad classes, it is particularly useful in crop plants, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum, and sugarcane. The present invention may also be used in conjunction with non-crop plants, including Arabidopsis thaliana.
The expression system of the present invention can be used to transform virtually any crop plant cell under suitable conditions. Cells transformed in accordance with the present invention can be grown in vitro in a suitable medium to confer protection against pesticide phytotoxicity. This protein can then be harvested or recovered by conventional purification techniques. The isolated protein can be applied to plants (e.g., by spraying) as a topical application to impart protection against pesticide phytotoxicity. Alternatively, transformed cells can be regenerated into whole plants such that this protein imparts protection against pesticide phytotoxicity to the intact transgenic plants. In either case, the plant cells transformed with the recombinant DNA expression system of the present invention are grown and caused to express that DNA in the cells to confer protection against pesticide phytotoxicity on them.
One technique of transforming plants with the DNA molecule in accordance with the present invention is by contacting the tissue of such plants with an inoculum of a bacteria transformed with a vector comprising a gene in accordance with the present invention which confers pesticide resistance. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25°-28°C
Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens (e.g., strains LBA4404 or EHA105) is particularly useful due to its well-known ability to transform plants. Another approach to transforming plant cells with a gene which confers protection against pesticide phytotoxicity involves propelling inert or biologically active particles at plant tissues cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792 all to Sanford et al., which are hereby incorporated by reference.
As noted above, in addition to having protection against pesticide phytotoxicity, plants transformed with a gene encoding for the cytochrome P450lpr polypeptide will be useful in killing insects when this polypeptide is produced.
The following examples illustrate, but are not intended to limit, the present invention.
PAC StrainThe LPR strain of the housefly was originally collected in 1982 from a dairy in New York, and selected with permethrin for 22 generations. The LPR attained to a high and stable level of resistance to pyrethroid insecticides with a phenoxybenzyl alcohol moiety (e.g. 6,000-fold permethrin resistance and >10,000-fold deltamethrin resistance). See J. G. Scott, et al., "Mechanisms Responsible for High Levels of Permethrin Resistance in the House Fly," Pestic. Sci., 17:195-206 (1986), which is hereby incorporated by reference. The flies were reared in a standard medium, and 0 day old adults were Used without further treatment for RNA extraction.
PAC Oligopeptide SequencingThe cytochrome P450lpr protein was purified from microsome of adult abdomens by HPLC, G. D. Wheelock, et al., "Simultaneous Purification of a Cytochrome P-450 and Cytochrome b5 from the House Fly Musca domestica L.", Insect Biochem., 19:481-489 (1989), which is hereby incorporated by reference, as a single immunoreactive protein. The isolated protein was cleaved with trypsin, and the resulting oligopeptides were sequenced on an Applied Biosystems, Model 470. Three of the 4 determined polypeptide sequences were utilized for degenerate oligonucleotide designation.
PAC cDNA AmplificationRNA was extracted from 15 abdomens by the guanidium isothiocyanate-hot phenol method. Poly(A)-RNA was screened by a Oligotex® Qiagen, Chatsworth, Calif. batch treatment method. The first-strand cDNA was synthesized with Molony murine leukemia virus ("M-MLV") reverse transcriptase (Perkin-Elmer, Norwalk, Conn.), 1 μg of poly(A)-enriched RNA, and an antisense primer or a 5'-anchored oligo(dT)16 primer, at 42°C for 1 hr. This step was followed by heat-inactivation and polymerase chain reaction ("PCR") with 0.2 mM each of a primer set in 100 μl reaction volume. Each reaction cycle (95°C for 0.5 min, 55°C for 1 min, and 72°C for 2 min) was repeated 25-30 times and followed by the final elongation step (72°C for 7 min).
PAC Genomic DNA AmplificationDNA was extracted from adult flies by proteinase K and phenol-chlorform treatments. The precipitated nucleic acids were treated with RNase A, and the initial extraction steps were repeated. One microgram DNA was used as a PCR template in a 100 μl reaction volume.
PAC Terminal Transferase ReactionThe first-strand cDNA synthesized with M-MLV reverse transcriptase was treated with phenol-chlorform and precipitated 4 times with Ethachinmate (an acrylamide DNA-carrier, Nippon Gene, Tokyo, Japan) by ethanol precipitation. dATPs were tailed to the 3' end of first-strand cDNA with terminal deoxynucleotidyl transferase (GIBCO BRL, Gaithersburg, Md.) in the condition described by the manufacturer. The cDNA was extracted by phenol-chlorform treatment and precipitated.
PAC DNA SequencingThe first PCR product was separated by agarose gel electrophoresis. A DNA band was extracted from the gel, purified by QIAEX® matrix protocol (Qiagen, Chatsworth, Calif.) and used as a template for a second PCR step with the same or internal primers. To the second PCR product, concentrated through ethanol precipitation, the same gel extraction procedures were applied. Double-stranded PCR product was directly sequenced by the dideoxynucleotide chain termination method with [@-35S]thiodATP and SEQUENASE® kit (United States Biochemical Corp., Cleveland, Ohio) according to the manufacturers protocol, which is hereby incorporated by reference, except for, the addition of dimethylsulfoxide at a final concentration of 10% (v/v) to the reaction solution, and DNA denaturation at 95°C for 3 min followed by quenching on dry ice.
PAC Southern HybridizationGenomic DNA fragments after digestion with EcoRI were separated by agarose gel electrophoresis, and blotted on nylon membrane. Pure PCR product from P450lpr cDNA coding region was labelled with [@-32P]dCTP by random priming method, and hybridized on the membrane with sheared salmon DNA in QUICK HYB® solution (Strategene, LaJolla, Calif.). Hybridization and washing followed high stringency condition.
PAC In Vitro Cloning StrategiesTo obtain cDNA clones which covers coding region, cDNA was synthesized and amplified by reverse transcription-mediated polymerase chain reaction (RT-PCR), separated into 3 parts. The sequences of the key primers used for RT-PCR are shown in Table 1, while their based amino acid sequences and the priming sites are mapped in FIG. 1.
| TABLE 1 |
| __________________________________________________________________________ |
| Oligonculeotides Used for cDNA Synthesis and Amplification |
| Corresponding |
| Oligonucleotide polypeptide |
| Names |
| Sequences sequences |
| __________________________________________________________________________ |
| S1'* |
| 5'-AC(CG)(TC)T(GC)TA(TC)AT(TC)TT(TC)GCCAA-3' |
| TLYIFAK |
| (SEQ. ID. No. 8) (SEQ. ID. No. 13) |
| AS3# |
| 5'-(TC)TG(AG)CC(TC)TCCAT(CATG)AC(AG)AA-3' |
| FVMEGQ |
| (SEQ. ID. No. 9) (SEQ. ID. No. 14) |
| AS4# |
| 5'-TC(ATC)GG(TC)TG(CTAG)GG(AG)AA(AG)TA(TC)TG-3' |
| QYFPQP |
| (SEQ. ID. No. 10) (SEQ. ID. No. 15) |
| C2 5'- TAATACGACTCACTATAGGGAGA-3' |
| (SEQ. ID. No. 11) |
| C2PT |
| 5'- TAATACGACTCACTATAGGGAGATTTTTTTTTTTTTTTT-3' |
| (SEQ. ID. No. 12) |
| __________________________________________________________________________ |
| Nucleotides in parentheses present mixed incorporation. |
| *Sense primer. |
| #Antisense primers. |
First, 2 overlapping internal sequences from coding region were amplified with the degenerate primers based on P450lpr polypeptide sequences (FIG. 1A); the longer PCR product of 1.4 kilobase pairs ("kbp") was obtained with S1 and AS4 primer set; the shorter one was obtained with S1 and AS3. Here we denote that a sense primer S1 is designed from N-terminal sequence. A serial sequence of 1.3 kb was obtained from these products followed by subsequent primer walking. Second, a cDNA sequence encoding C-terminal region was amplified with the first-strand cDNA primed by the 5'-anchored poly(dT) oligonucleotide, C2PT (FIG. 1B); this cDNA was amplified by single side-nested PCR with coding region specific sense primers (upstream from AS4, sequences not shown) and the anchor specific C2 primer. A sequence of 160 bases was obtained with this product. Third, a cDNA sequence encoding N-terminus was amplified with the first-strand cDNA that was primed by an antisense primer (downstream from S1, sequence not shown) and tailed at the 3' end by poly-(dA) by terminal deoxynucleotidyl transferase reaction (FIG. 1C). This cDNA was amplified following the analogous nested PCR and a sequence of 530 bases was determined.
PAC Nucleotide SequenceA serial sequence of 1816 bases (FIG. 2) was obtained from the 3 in vitro cDNA clones. This sequence has an open reading frame of 1548 bases, coding a 516 residues (Mr 59,182). This agrees with a molecular mass of the purified protein. See G. D. Wheelock, et al., "Simultaneous Purification of a Cytochrome P-450 and Cytochrome b5 from the House Fly Musca domestica L", Insect Biochem. 19:481-489 (1989), which is hereby incorporated by reference. Putative polyadenylation signals, AUUUA (SEQ. ID. No. 16) and AAUAAA (SEQ. ID. No. 17), appear twice within the 3'-untranslated region, although no poly(A) was observed.
Three cDNA probes upstream and/or downstream from an unique EcoRI site in the obtained cDNA sequence (FIGS. 1 and 2) were used for orienting the genomic DNA fragments from LPR flies. Two EcoRI DNA fragments of 2.2 and 3.3 kbp, hybridized with upstream and downstream cDNA probes, respectively, and they were oriented in this order (FIG. 3). This result also shows that the DNA sequence obtained was derived from an identical gene sequence, as well as the involvement of experimentally obtained 4 polypeptide sequences in the deduced protein sequence (FIG. 2).
Intron insertion was analyzed by differential size screening of paired PCR products which were derived from cDNA and genomic DNA templates with common primer sets, followed by the subsequent sequencing of the genomic DNA-PCR products (FIG. 1D). A total of 5 pairs of PCR products in screening covers a 5'-untranslated region and most of the coding region (cDNA bases -88 to 1544) with serial overlaps. Three introns with 74, 66, and 64 bases have split within the coding sequence in this order (for insertion site, see FIG. 2) and they were tentatively numbered as intron 1, and 3, respectively. ##STR4## Each sequence had a canonical 5'-GT and AG-3' terminal bases.
PAC Comparison with Other P450 SequencesThe cytochrome P450lpr sequence involves a FXXGXXXC sequence (SEQ. ID. No. 21) near the N-terminus (FIG. 2). In SEQ. ID. No. 21, X can be any amino acid. This sequence is invariant in P450 super gene family members and the best key for characterizing P450 sequences, and Cys-461 in this sequence is known as the fifth ligand to heme. Also Gly-455, Arg-459, Ala-463, Gly-467, and Ans-470 in or C-distal from this invariant residue appear to be major or already identified residues at the conserved positions within heme-binding region. The N-terminal region of the lpr sequence is highly hydrophobic over first 19 residues that agrees with a common feature of N-terminal region of microsomal P450s as an membrane-anchor signal. The other conserved or possible functional regions involved in the lpr are discussed infra.
The P450lpr exhibits the greatest regional similarity index with Drosophila melanogaster CYP6A2 and black swallowtail butterfly (Papilio polyxenes) CYP6B1, and also exhibits a high index with housefly CYP6A1 as well as with P450III family mammalian proteins. To close up conserved amino acids in cytochrome P450lpr, an alignment was made with the cytochrome P450lpr and its similar 4 most similar sequences including the above 3 insect proteins and rat pcn1 (CYP3A1) (FIG. 4). First, multiple alignment among cytochrome P450lpr (SEQ. ID. No. 2), 6A1 (SEQ. ID. No. 22), 6A2 (SEQ. ID. No. 23), and 6B1 (SEQ. ID. No. 24) was made. This alignment was then overlaid onto the pcn1 sequence. The obtained alignment had identical residues and the residues shared among 4 sequences at 59 and 60 positions, respectively (FIG. 4). Based on this alignment, positional identity is presented in Table 2 using a total alignment window (positions 1-547).
| TABLE 2 |
| ______________________________________ |
| Percent Amino Acid Identity of P450s in Multiple Alignment |
| pcn1 6A1 6A2 6B1 1pr |
| ______________________________________ |
| Rat pcn1 -- 30.7 25.2 23.8 21.4 |
| H.fly 6A1 30.5 -- 37.8 27.1 23.8 |
| F.fly 6A2 26.0 38.3 -- 30.0 21.2 |
| Butterfly |
| 6B1 23.9 26.8 30.1 -- 26.3 |
| H.fly P4501pr |
| 21.9 24.5 21.7 27.0 -- |
| ______________________________________ |
| The values in the upper right and lower left columns are based on a total |
| aligment window (alignment positions 1-547) and a partial alignment windo |
| (33-547), respectively. |
Amino acid identity of cytochrome P450lpr is 26.3, 23.8, 21.4, and 21.2% with 6B1, 6A1, pcn1 (CYP3A1) (SEQ. ID. No. 25), and 6A2, respectively. Higher identities are presented in sequence pairs of 6A1 and 6A2 (37.8%), 6A1 and pcn1 (30.7%), and 6A2 and 6B1 (30.0%). Also amino acid identity was calculated based on a partial alignment window (positions 33-547) to eliminate vulnerable alignment within the N-terminal region due to repetitive appearance of hydrophobic residues such as neu, although similarity tendency was essentially the same as the above results (Table 2). The hydrophobicity profile was quite similar among these 5 sequences, suggesting similarity in higher order structure among them.
The housefly P450lpr has several typical conserved regions as a microsomal P450 protein, as well as heme-binding region and highly hydrophobic N-terminal region. Possible functional regions in P450lpr are revealed in connection with the alignment from its most similar sequences (FIG. 4). (i) The invariant charged residues of Glu-380 and Arg-383 in lpr are involved in Helix K region in P450 cam. Tyl-373 in lpr is also conserved among animal P450s together with an alternate Leu at this position, while lpr and members of P450 family VI and III present variable residues at the lpr Leu-376 position instead of conserved Ala.
A typical sequence of P450 aromatic region appears with 2 invariant Pro residues and 3 aromatic residues within FXXPXXYXPXRF (SEQ. ID. No. 26) frame (residues 428-439) in lpr. In any SEQ. ID. No. 26, X is any amino acid. Phe-439 in this frame is a characteristic residue within microsomal P450s. Involvement of Helix K and aromatic regions in interaction with an electron-donor protein by intermolecular ion-pairing has been suggested.
Positions 126, 392, and 459 in cytochrome P450lpr are occupied by basic residues Arg. Conserved basic residues at these positions in animal P450s as well as P450cam may form ion pairs with heme propionate to incorporate essentially the same tertiary structure as P450cam. Thr-322 in cytochrome P450lpr appears to be an invariant residue. The Helix I region in P450cam involves this residue and may play a crucial role in catalytic function when in contact with the heme surface to bind oxygen and substrate molecules. A conserved acidic position just N-proximal to this Thr (Glu-321 in lpr) may form an internal ion pair with a basic residue located in the highly variable region. A basic residue Lys-343 and an acidic residue Asp-349 surrounding highly conserved Glu-347 in cytochrome P450lpr agree with a conserved charged frame among microsomal P450s. The corresponding Glu in P450cam is involved in Helix J region. This region can be incorporated together with Helix K region in the specific intermolecular interaction with a component of the electron-transfer system.
P450lpr protein sequence exhibits similarity to the members of P450 family VI and III. Lpr specifically catalyses pyrethroid insecticides with phenoxybenzyl alcohol moiety. Black swallow tail CYP6B1 presenting the highest similarity (26.2%) to cytochrome P450lpr metabolizes xanthotoxin in plant feeding of caterpillar. See Cohen, et al., M. Cohen, et al., "A Host-Inducable Cytochrome P450 from a Host Specific Caterpillar Molecule Cloning and Evolution," Proc. Nat. Acad. Sci. USA, 89:10920-24 (1992). Housefly 6A1 and D. melanogaster 6A2 are involved in detoxification of DDT and organophosphate insecticides, respectively, by their elevated expression in each resistant strain. See J. G. Scott, "The Cytochrome P450 Microsomal Monooxygenases of Insects: Recent Advances," Rev. Pestic. Toxicol., Vol. 2, R. M. Roe et. al., editors (1993), which is hereby incorporated by reference. Rat pcn1 selectively metabolizes testosterone. It is elusive to correlate the protein structures with specific metabolic roles to date. Comparison of cytochrome P450lpr sequence with its similar 4 sequences reveals apparently excessive stretches in cytochrome P450lpr within the residues 169-163, 218-228, 295-307, and 355-360 (FIG. 4), and it is difficult to achieve alignment around these stretches. Two of them (residues 218-228 and 295-307) correspond to hyper variable domains in P450 supergene family members; the former stretch corresponds to a substrate binding region (spanning 175-207) of P450cam; and the latter corresponds to a postulated substrate binding region in mammalian P450s which can be localized in N-proximal to a substrate binding residue (within 243-252) of P450cam. Whether there are essential residues or local arrangements responsible for specific catalytic reactions in these stretches remains to be elucidated.
Three intron sequences were found from the coding region in cytochrome P450lpr genomic DNA, and they are inserted at sites within the corresponding codons of Thr-176, Glu-380, and Ala-463 (FIG. 2). The cytochrome P450lpr intron sites were compared with rat c (CYP1A1), rat b (CYP2A1), human scc (CYP11A1), and human c21b (CYP21A2) genes. Each of the mammalian genes was picked up as a representative member of the P450 family in which the gene structure is known. The codons corresponding to lpr intron insertion sites are Val-196, Glu-377, and Glu-463 in rat c genes. These positions are apparently different from that involved in the 4 mammalian genes described above. It seems difficult to explain a postulated idea that P450 gene families have diverged with deleting the introns involved in an ancestral gene sequence. Unfortunately, no other intron information is available from most similar P450III members and P450VI insect members to date, although an intron is not involved in Drosophila CYP6A2 gene structure. Evolutionary relationships among lpr and other barely similar P450s might be clarified from further intron information.
With the obtained cytochrome P450lpr cDNA sequence, there are several themes to be investigated in relation with genetic mechanism of elevated pyrethroid detoxification activity of lpr in insecticide resistant flies. First, is catalytic change in the molecule responsible for elevated detoxification of pyrethroids? As introduced, microsomes from pyrethroid resistant LPR strain of flies specifically detoxify deltamethrin. Several genetic polymorphisms responsible for amino acid substitution are identified in cDNA sequences from LPR and a susceptible strain. Second, whether an elevated expression of lpr is due to a difference in the copy number of the structure gene or transcriptional regulation. Cytochrome P450lpr protein is considerably overproduced in LPR flies. Esterase gene amplifications involved in organophosphate resistance in aphid and mosquito (J. G. Scott, "Insecticide Resistance in Insects," Handbook of Pest Management, P. Pimentel (ed.), CRC Press, pp. 663-77 (1991) have been well exemplified cases as the former possibility. However, in the case of cytochrome P450lpr, overproduction in the LPR strain, at least the latter genetic mechanism should be working. Chromosomal linkage of the cytochrome P450lpr gene is already determined using the internal gene sequence polymorphism; the genetic factors which remarkably elevate the level of cytochrome P450lpr protein expression is localized not only within the same linkage as the cytochrome P450lpr gene but also in another chromosome by an immunoquantification assay. Thirdly, the DNA element and the factor which explains the difference in genetic inducibility of cytochrome P450lpr gene must be investigated. Interestingly, the cytochrome P450lpr proteins in insecticide susceptible stains is effectively inducible by phenobarbital with adult flies, while the cytochrome P450lpr protein in LPR strain is constitutively expressed over adult ages and the same induction treatment is not effective. See J. G. Scott, "The Cytochrome P450 Microsomal Monooxygenases of Insects: Recent Advances," Rev. Pestic. Toxicol., Vol. 2, R. M. Roe et. al., editors (1993).
PAC Comparison with Nucleotide and Amino Acid Sequences for CYP6D1 Allele for Susceptible FliesThe nucleotide sequence and deduced amino acid sequence for CS strain house flies susceptible to insecticides was determined in substantially the same way as described in Examples 2-9, starting with susceptible flies. The nucleotide sequence for susceptible house flies (SEQ. ID. No. 27) is: ##STR5## The amino acid sequence (SEQ. ID. No. 28) for susceptible house flies is:
| ______________________________________ |
| Met Leu Leu Leu Leu Leu Leu Ile Val Val |
| Thr Thr Leu Tyr Ile Phe Ala Lys Leu His |
| Tyr Thr Lys Trp Glu Arg Leu Gly Phe Glu |
| Ser Asp Lys Ala Thr Ile Pro Leu Gly Ser |
| Met Ala Lys Val Phe His Lys Glu Arg Pro |
| Phe Gly Leu Val Met Ser Asp Ile Tyr Asp |
| Lys Cys His Glu Lys Val Val Gly Ile Tyr |
| Leu Phe Phe Lys Pro Ala Leu Leu Val Arg |
| Asp Ala Glu Leu Ala Arg Gln Ile Leu Thr |
| Thr Asp Phe Asn Ser Phe His Asp Arg Gly |
| Leu Tyr Val Asp Glu Lys Asn Asp Pro Met |
| Ser Ala Asn Leu Phe Val Met Glu Gly Gln |
| Ser Trp Arg Thr Leu Arg Met Lys Leu Ala |
| Pro Ser Phe Ser Ser Gly Lys Leu Lys Gly |
| Met Phe Glu Thr Val Asp Asp Val Ala Asp |
| Lys Leu Ile Asn His Leu Asn Glu Arg Leu |
| Lys Asp Gly Gln Thr His Val Leu Glu Ile |
| Lys Ser Ile Leu Thr Thr Tyr Ala Val Asp |
| Ile Ile Gly Ser Val Ile Phe Gly Leu Glu |
| Ile Asp Ser Phe Thr His Pro Asp Asn Glu |
| Phe Arg Val Leu Ser Asp Arg Leu Phe Asn |
| Pro Lys Lys Ser Thr Met Leu Glu Arg Phe |
| Arg Asn Leu Ser Thr Phe Met Cys Pro Pro |
| Leu Ala Lys Leu Leu Ser Arg Leu Gly Ala |
| Lys Asp Pro Ile Thr Tyr Arg Leu Arg Asp |
| Ile Val Lys Arg Thr Ile Glu Phe Arg Glu |
| Glu Lys Gly Val Val Arg Lys Asp Leu Leu |
| Gln Leu Phe Ile Gln Leu Arg Asn Thr Gly |
| Lys Ile Ser Asp Asp Asn Asp Lys Leu Trp |
| His Asp Val Glu Ser Thr Ala Glu Asn Leu |
| Lys Ala Met Ser Ile Asp Met Ile Ala Ser |
| Asn Ser Phe Leu Phe Tyr Ile Ala Gly Ser |
| Glu Thr Thr Ala Ala Thr Thr Ser Phe Thr |
| Ile Tyr Glu Leu Ala Met Tyr Pro Glu Ile |
| Leu Lys Lys Ala Gln Ser Glu Val Asp Glu |
| Cys Leu Gln Arg His Gly Leu Lys Pro Gln |
| Gly Arg Leu Thr Tyr Glu Ala Ile Gln Asp |
| Met Lys Tyr Leu Asp Leu Cys Val Met Glu |
| Thr Thr Arg Lys Tyr Pro Gly Leu Pro Phe |
| Leu Asn Arg Lys Cys Thr Gln Asp Phe Gln |
| Val Pro Asp Thr Lys Leu Thr Ile Pro Lys |
| Glu Thr Gly Ile Ile Ile Ser Leu Leu Gly |
| Ile His Thr His Pro Gln Tyr Phe Pro Gln |
| Pro Glu Asp Tyr Arg Pro Glu Arg Phe Ala |
| Asp Glu Ser Lys Asp Tyr Asp Pro Ala Ala |
| Tyr Met Pro Phe Gly Glu Gly Pro Arg His |
| Cys Ile Ala Gln Arg Met Gly Val Met Asn |
| Ser Lys Val Ala Leu Ala Lys Ile Leu Ala |
| Asn Phe Asn Ile Gln Pro Met Pro Arg Gln |
| Glu Val Glu Phe Lys Phe His Ser Ala Pro |
| Val Leu Val Pro Val Asn Gly Leu Asn Val |
| Gly Leu Ser Lys Arg Trp TGA |
| ______________________________________ |
As shown in FIG. 5, a comparison of the amino acid sequence for cytochrome P450lpr (SEQ. ID. No. 2), identified as LPR in FIG. 5, and the corresponding amino acid sequence for susceptible house flies (SEQ. ID. No. 28), identified as CS in FIG. 5, demonstrates that these sequences are substantially similar except that amino acid 220 is isoleucine in the former and phenylalanine in the latter, while amino acid 469 is isoleucine in the former and methionine in the latter. These differences are highlighted by enclosure in rectangles in FIG. 5. In FIGS. 6A-D, a comparison of the nucleotide sequence for cytochrome P450lpr (SEQ. ID. No. 1), identified as LPR in FIGS. 6A-D, and the corresponding nucleotide sequence for susceptible house flies (SEQ. ID. No. 29), identified as CS in FIGS. 6A-D, is made. This demonstrates that there are a number of differences in the nucleotides even though most of these differences do not change the encoded amino acids. The two underlined nucleotides in FIGS. 6A-D are the only differences between the CS allele and LPR allele that result in amino acid changes in the protein. Whether or not the change in the two amino acids between CS and LPR alters the catalytic activity of the protein is unknown.
Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.
| __________________________________________________________________________ |
| SEQUENCE LISTING |
| (1) GENERAL INFORMATION: |
| (iii) NUMBER OF SEQUENCES: 29 |
| (2) INFORMATION FOR SEQ ID NO:1: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 1551 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: double |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA (cDNA) |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: Learn-PyR |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: |
| ATGTTGTTATTACTGCTGCTGATTGTGGTGACGACCCTCTATATCTTTGCCAAACTCCAT60 |
| TATACGAAATGGGAACGTTTGGGTTTCGAATCGGATAAGGCCACCATACCCCTGGGCTCA120 |
| ATGGCAAAGGTATTCCACAAGGAACGGCCATTTGGCCTGGTTATGTCCGACATATATGAC180 |
| AAATGCCACGAGAAGGTGGTGGGCATTTATTTGTTCTTCAAGCCGGCCCTACTGGTACGT240 |
| GATGCCGAATTGGCGAGACAAATTTTGACCACGGATTTTAATAGCTTCCACGATCGTGGC300 |
| CTCTATGTGGATGAGAAAAATGATCCAATGTCGGCGAATCTTTTCGTGATGGAGGGTCAA360 |
| TCATGGCGTACGCTGAGAATGAAATTGGCCCCCTCGTTTTCGTCGGGTAAACTCAAGGGG420 |
| ATGTTCGAAACGGTCGATGATGTGGCGGATAAATTAATAAATCACTTGAATGAGCGCTTG480 |
| AAGGATGGCCAGACGCATGTTTTGGAAATCAAGAGTATTTTGACCACCTATGCTGTCGAC540 |
| ATCATTGGTTCGGTGATATTCGGCCTGGAAATCGATAGTTTCACCCATCCGGACAATGAA600 |
| TTTCGTGTCTTAAGTGATCGTCTATTTAACCCAAAGAAGTCGACAATGTTGGAGAGAATT660 |
| CGCAATTTATCAACCTTTATGTGTCCACCACTTGCCAAACTCTTGTCGCGCCTTGGTGCC720 |
| AAGGATCCGATAACATATCGCCTGCGCGACATCGTGAAACGGACGATAGAATTTCGCGAA780 |
| GAAAAGGGCGTTGTACGCAAAGATCTTCTCCAGCTATTTATACAACTCAGAAATACTGGA840 |
| AAAATTTCCGATGACAATGACAAGCTATGGCATGACGTTGAGTCGACGGCGGAAAATCTC900 |
| AAAGCCATGTCTATCGATATGATTGCCTCCAATTCATTCCTATTCTATATTGCCGGTTCG960 |
| GAAACAACGGCGGCCACAACATCATTTACCATCTATGAATTGGCCATGTATCCGGAAATT1020 |
| TTGAAAAAGGCCCAATCTGAGGTGGATGAGTGCCTGCAAAGGCATGGTCTCAAGCCGCAG1080 |
| GGACGGCTGACATATGAGGCAATACAGGATATGAAATATTTGGATTTGTGTGTTATGGAA1140 |
| ACCACCCGCAAATACCCCGGCCTGCCGTTTTTGAATCGCAAATGCACTCAGGATTTCCAA1200 |
| GTACCCGACACAAAACTGACCATACCAAAGGAAACGGGAATTATCATCTCCTTGTTGGGC1260 |
| ATCCATAGAGACCCACAGTATTTCCCCCAACCCGAGGATTATAGGCCAGAACGCTTTGCC1320 |
| GATGAGAGCAAGGATTATGATCCAGCGGCATATATGCCTTTTGGAGAGGGTCCAAGGCAT1380 |
| TGTATTGCTCAACGCATGGGCGTTATCAATTCCAAGGTAGCCTTGGCCAAAATATTGGCC1440 |
| AATTTTAATATTCAACCAATGCCCCGCCAAGAAGTTGAGTTCAAATTCCATTCAGCTCCT1500 |
| GTTCTGGTGCCAGTAAATGGTCTCAATGTGGGCCTGTCGAAGAGGTGGTGA1551 |
| (2) INFORMATION FOR SEQ ID NO:2: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 517 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: |
| MetLeuLeuLeuLeuLeuLeuIleValValThrThrLeuTyrIlePhe |
| 151015 |
| AlaLysLeuHisTyrThrLysTrpGluArgLeuGlyPheGluSerAsp |
| 202530 |
| LysAlaThrIleProLeuGlySerMetAlaLysValPheHisLysGlu |
| 354045 |
| ArgProPheGlyLeuValMetSerAspIleTyrAspLysCysHisGlu |
| 505560 |
| LysValValGlyIleTyrLeuPhePheLysProAlaLeuLeuValArg |
| 65707580 |
| AspAlaGluLeuAlaArgGlnIleLeuThrThrAspPheAsnSerPhe |
| 859095 |
| HisAspArgGlyLeuTyrValAspGluLysAsnAspProMetSerAla |
| 100105110 |
| AsnLeuPheValMetGluGlyGlnSerTrpArgThrLeuArgMetLys |
| 115120125 |
| LeuAlaProSerPheSerSerGlyLysLeuLysGlyMetPheGluThr |
| 130135140 |
| ValAspAspValAlaAspLysLeuIleAsnHisLeuAsnGluArgLeu |
| 145150155160 |
| LysAspGlyGlnThrHisValLeuGluIleLysSerIleLeuThrThr |
| 165170175 |
| TyrAlaValAspIleIleGlySerValIlePheGlyLeuGluIleAsp |
| 180185190 |
| SerPheThrHisProAspAsnGluPheArgValLeuSerAspArgLeu |
| 195200205 |
| PheAsnProLysLysSerThrMetLeuGluArgIleArgAsnLeuSer |
| 210215220 |
| ThrPheMetCysProProLeuAlaLysLeuLeuSerArgLeuGlyAla |
| 225230235240 |
| LysAspProIleThrTyrArgLeuArgAspIleValLysArgThrIle |
| 245250255 |
| GluPheArgGluGluLysGlyValValArgLysAspLeuLeuGlnLeu |
| 260265270 |
| PheIleGlnLeuArgAsnThrGlyLysIleSerAspAspAsnAspLys |
| 275280285 |
| LeuTrpHisAspValGluSerThrAlaGluAsnLeuLysAlaMetSer |
| 290295300 |
| IleAspMetIleAlaSerAsnSerPheLeuPheTyrIleAlaGlySer |
| 305310315320 |
| GluThrThrAlaAlaThrThrSerPheThrIleTyrGluLeuAlaMet |
| 325330335 |
| TyrProGluIleLeuLysLysAlaGlnSerGluValAspGluCysLeu |
| 340345350 |
| GlnArgHisGlyLeuLysProGlnGlyArgLeuThrTyrGluAlaIle |
| 355360365 |
| GlnAspMetLysTyrLeuAspLeuCysValMetGluThrThrArgLys |
| 370375380 |
| TyrProGlyLeuProPheLeuAsnArgLysCysThrGlnAspPheGln |
| 385390395400 |
| ValProAspThrLysLeuThrIleProLysGluThrGlyIleIleIle |
| 405410415 |
| SerLeuLeuGlyIleHisArgAspProGlnTyrPheProGlnProGlu |
| 420425430 |
| AspTyrArgProGluArgPheAlaAspGluSerLysAspTyrAspPro |
| 435440445 |
| AlaAlaTyrMetProPheGlyGluGlyProArgHisCysIleAlaGln |
| 450455460 |
| ArgMetGlyValIleAsnSerLysValAlaLeuAlaLysIleLeuAla |
| 465470475480 |
| AsnPheAsnIleGlnProMetProArgGlnGluValGluPheLysPhe |
| 485490495 |
| HisSerAlaProValLeuValProValAsnGlyLeuAsnValGlyLeu |
| 500505510 |
| SerLysArgTrpXaa |
| 515 |
| (2) INFORMATION FOR SEQ ID NO:3: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 19 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: |
| MetLeuLeuLeuLeuLeuLeuIleValValThrThrLeuTyrIlePhe |
| 151015 |
| AlaLysLeu |
| (2) INFORMATION FOR SEQ ID NO:4: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 21 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: |
| GlyLeuTyrValAspGluLysAsnAspProMetSerAlaAsnLeuPhe |
| 151015 |
| ValMetGluGlyGln |
| 20 |
| (2) INFORMATION FOR SEQ ID NO:5: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 6 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: |
| AspAspAsnAspLysLeu |
| 15 |
| (2) INFORMATION FOR SEQ ID NO:6: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 12 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: |
| GluThrGlyIleIleIleSerLeuLeuGlyIleHis |
| 1510 |
| (2) INFORMATION FOR SEQ ID NO:7: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 13 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: |
| ProGlnTyrPheProGlnProGluAspTyrArgProGlu |
| 1510 |
| (2) INFORMATION FOR SEQ ID NO:8: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 26 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: |
| ACCGTCTGCTATCATTCTTTCGCCAA26 |
| (2) INFORMATION FOR SEQ ID NO:9: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 25 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: |
| TCTGAGCCTCTCCATCATGACAGAA25 |
| (2) INFORMATION FOR SEQ ID NO:10: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 29 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: |
| TCATCGGTCTGCTAGGGAGAAAGTATCTG29 |
| (2) INFORMATION FOR SEQ ID NO:11: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 23 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: |
| TAATACGACTCACTATAGGGAGA23 |
| (2) INFORMATION FOR SEQ ID NO:12: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 39 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: |
| TAATACGACTCACTATAGGGAGATTTTTTTTTTTTTTTT39 |
| (2) INFORMATION FOR SEQ ID NO:13: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 7 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: |
| ThrLeuTyrIlePheAlaLys |
| 15 |
| (2) INFORMATION FOR SEQ ID NO:14: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 6 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: Learn-PyR |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: |
| PheValMetGluGlyGln |
| 15 |
| (2) INFORMATION FOR SEQ ID NO:15: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 6 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: Learn-PyR |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: |
| GlnTyrPheProGlnPro |
| 15 |
| (2) INFORMATION FOR SEQ ID NO:16: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 2 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: double |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA (genomic) |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: Learn-PyR |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: |
| AA2 |
| (2) INFORMATION FOR SEQ ID NO:17: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 5 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA (genomic) |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: Learn-PyR |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: |
| AAAAA5 |
| (2) INFORMATION FOR SEQ ID NO:18: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 74 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: double |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA (genomic) |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: Learn-PyR |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: |
| GTAAGTACTCATCGTTGAGAGAATTGTAAGAAGTTTTGAATTTTACTTTTAATAAAATGT60 |
| TCTTCTTCCCCCAG74 |
| (2) INFORMATION FOR SEQ ID NO:19: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 66 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: double |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA (genomic) |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: Learn-PyR |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: |
| GTAAGAGGGGAAATTTTGAAATTGTTTTTTTTTTTATTTTCTAATTATTGCATGTTTTTG60 |
| TTGTAG66 |
| (2) INFORMATION FOR SEQ ID NO:20: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 64 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: double |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA (genomic) |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: Learn-PyR |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: |
| GTGAGATGTTGAAAGGGGAGCTTCATTAAATTCTGAATATTAATTTTGTATTTTTTTTCC60 |
| ACAG64 |
| (2) INFORMATION FOR SEQ ID NO:21: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 8 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: Learn-PyR |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: |
| PheXaaXaaGlyXaaXaaXaaCys |
| 15 |
| (2) INFORMATION FOR SEQ ID NO:22: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 507 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: Rutgers |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 5 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: |
| MetAspPheGlySerPheLeuLeuTyrAlaLeuGlyValLeuAlaSer |
| 151015 |
| LeuAlaLeuTyrPheValArgTrpAsnPheGlyTyrTrpLysArgArg |
| 202530 |
| GlyIleProHisGluGluProHisLeuValMetGlyAsnValLysGly |
| 354045 |
| LeuArgSerLysTyrHisIleGlyGluIleIleAlaAspTyrTyrArg |
| 505560 |
| LysPheLysGlySerGlyProPheAlaGlyIlePheLeuGlyHisLys |
| 65707580 |
| ProAlaAlaValValLeuAspLysGluLeuArgLysArgValLeuIle |
| 859095 |
| LysAspPheSerAsnPheAlaAsnArgGlyLeuTyrTyrAsnGluLys |
| 100105110 |
| AspAspProLeuThrGlyHisLeuValMetValGluGlyGluLysTrp |
| 115120125 |
| ArgSerLeuArgThrLysLeuSerProThrPheThrAlaGlyLysMet |
| 130135140 |
| LysTyrMetTyrAsnThrValLeuGluValGlyGlnArgLeuLeuGlu |
| 145150155160 |
| ValMetTyrGluLysLeuGluValSerSerGluLeuAspMetArgAsp |
| 165170175 |
| IleLeuAlaArgPheAsnThrAspValIleGlySerValAlaPheGly |
| 180185190 |
| IleGluCysAsnSerLeuArgAsnProHisAspArgPheLeuAlaMet |
| 195200205 |
| GlyArgLysSerIleGluValProArgHisAsnAlaLeuIleMetAla |
| 210215220 |
| PheIleAspSerPheProGluLeuSerArgLysLeuGlyMetArgVal |
| 225230235240 |
| LeuProGluAspValHisGlnPhePheMetSerSerIleLysGluThr |
| 245250255 |
| ValAspTyrArgGluLysAsnAsnIleArgArgAsnAspPheLeuAsp |
| 260265270 |
| LeuValLeuAspLeuLysAsnAsnProGluSerIleSerLysLeuGly |
| 275280285 |
| GlyLeuThrPheAsnGluLeuAlaAlaGlnValPheValPhePheLeu |
| 290295300 |
| GlyGlyPheGluThrSerSerSerThrMetGlyPheAlaLeuTyrGlu |
| 305310315320 |
| LeuAlaGlnAsnGlnGlnLeuGlnAspArgLeuArgGluGluValAsn |
| 325330335 |
| GluValPheAspGlnPheLysGluAspAsnIleSerTyrAspAlaLeu |
| 340345350 |
| MetAsnIleProTyrLeuAspGlnValLeuAsnGluThrLeuArgLys |
| 355360365 |
| TyrProValGlySerAlaLeuThrArgGlnThrLeuAsnAspTyrVal |
| 370375380 |
| ValProHisAsnProLysTyrValLeuProLysGlyThrLeuValPhe |
| 385390395400 |
| IleProValLeuGlyIleHisTyrAspProGluLeuTyrProAsnPro |
| 405410415 |
| GluGluPheAspProGluArgPheSerProGluMetValLysGlnArg |
| 420425430 |
| AspSerValAspTrpLeuGlyPheGlyAspGlyProArgAsnCysIle |
| 435440445 |
| GlyMetArgPheGlyLysMetGlnSerArgLeuGlyLeuAlaLeuVal |
| 450455460 |
| IleArgHisPheArgPheThrValCysSerArgThrAspIleProMet |
| 465470475480 |
| GlnIleAsnProGluSerLeuAlaTrpThrProLysAsnAsnLeuTyr |
| 485490495 |
| LeuAsnValGlnAlaIleArgLysLysIleLys |
| 500505 |
| (2) INFORMATION FOR SEQ ID NO:23: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 507 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: drosophila melanogaster |
| (B) STRAIN: |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: |
| MetPheValLeuIleTyrLeuLeuIleAlaIleSerSerLeuLeuAla |
| 151015 |
| TyrLeuTyrHisArgAsnPheAsnTyrTrpAsnArgArgGlyLeuPro |
| 202530 |
| HisAspAlaProHisProLeuTyrGlyAsnMetValGlyPheArgLys |
| 354045 |
| AsnArgValMetHisAspPhePheTyrAspTyrTyrAsnLysTyrArg |
| 505560 |
| LysSerGlyPheProPheValGlyPheTyrPheLeuHisLysProAla |
| 65707580 |
| AlaPheIleValAspThrGlnLeuAlaLysAsnIleLeuIleLysAsp |
| 859095 |
| PheSerAsnPheAlaAspArgGlyGlnPheHisAsnGlyArgAspAsp |
| 100105110 |
| ProLeuThrGlnHisLeuPheAsnLeuAspGlyLysLysTrpLysAsp |
| 115120125 |
| MetArgGlnArgLeuThrProThrPheThrSerGlyLysMetLysPhe |
| 130135140 |
| MetPheProThrValIleLysValSerGluGluPheValLysValIle |
| 145150155160 |
| ThrGluGlnValProAlaAlaGlnAsnGlyAlaValLeuGluIleLys |
| 165170175 |
| GluLeuMetAlaArgPheThrThrAspValIleGlyThrCysArgPhe |
| 180185190 |
| GlyIleGluCysAsnThrLeuArgThrProValSerAspPheArgThr |
| 195200205 |
| MetGlyGlnLysValPheThrAspMetArgHisGlyLysLeuLeuThr |
| 210215220 |
| MetPheValPheSerPheProLysLeuAlaSerArgLeuArgMetArg |
| 225230235240 |
| MetMetProGluAspValHisGlnPhePheMetArgLeuValAsnAsp |
| 245250255 |
| ThrIleAlaLeuArgGluArgGluAsnPheLysArgAsnAspPheMet |
| 260265270 |
| AsnLeuLeuIleGluLeuLysGlnLysGlySerSerPheThrLeuAsp |
| 275280285 |
| AsnGlyGluValIleGluGlyMetAspIleGlyGluLeuAlaAlaGln |
| 290295300 |
| ValPheValPheTyrValAlaGlyPheGluThrSerSerSerThrMet |
| 305310315320 |
| SerTyrCysLeuTyrGluLeuAlaGlnAsnGlnAspIleGlnAspArg |
| 325330335 |
| LeuArgAsnGluIleGlnThrValLeuGluGluGlnGluGlyGlnLeu |
| 340345350 |
| ThrTyrGluSerIleLysAlaMetThrTyrLeuAsnGlnValIleSer |
| 355360365 |
| GluThrLeuArgLeuTyrThrLeuValProHisLeuGluArgLysAla |
| 370375380 |
| LeuAsnAspTyrValValProGlyHisGluLysLeuValIleGluLys |
| 385390395400 |
| GlyThrGlnValIleIleProAlaCysAlaTyrHisArgAspGluAsp |
| 405410415 |
| LeuTyrProAsnProGluThrPheAspProGluArgPheSerProGlu |
| 420425430 |
| LysValAlaAlaArgGluSerValGluTrpLeuProPheGlyAspGly |
| 435440445 |
| ProArgAsnCysIleGlyMetArgPheGlyGlnMetGlnAlaArgIle |
| 450455460 |
| GlyLeuAlaGlnIleIleSerArgPheArgValSerValCysAspThr |
| 465470475480 |
| ThrGluIleProLeuLysTyrSerProMetSerIleValLeuGlyThr |
| 485490495 |
| ValGlyGlyIleTyrLeuArgValGluArgIle |
| 500505 |
| (2) INFORMATION FOR SEQ ID NO:24: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 498 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: papillio polyxnes |
| (B) STRAIN: |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: |
| MetLeuTyrLeuLeuAlaLeuValThrValLeuAlaGlyLeuLeuHis |
| 151015 |
| TyrTyrPheThrArgThrPheAsnTyrTrpLysLysArgAsnValAla |
| 202530 |
| GlyProLysProValProPhePheGlyAsnLeuLysAspSerValLeu |
| 354045 |
| ArgArgLysProGlnValMetValTyrLysSerIleTyrAspGluPhe |
| 505560 |
| ProAsnGluLysValLeuGlyIleTyrArgMetThrThrProSerVal |
| 65707580 |
| LeuLeuArgAspLeuAspIleIleLysHisValLeuIleLysAspPhe |
| 859095 |
| GluSerPheAlaAspArgGlyValGluPheSerLeuAspGlyLeuGly |
| 100105110 |
| AlaAsnIlePheHisAlaAspGlyAspArgTrpArgSerLeuArgAsn |
| 115120125 |
| ArgPheThrProLeuPheThrSerGlyLysLeuLysSerMetLeuPro |
| 130135140 |
| LeuMetSerGlnValGlyAspArgPheIleAsnSerIleAspGluVal |
| 145150155160 |
| SerGlnThrGlnProGluGlnSerIleHisAsnLeuValGlnLysPhe |
| 165170175 |
| ThrMetThrAsnIleAlaAlaCysValPheGlyLeuAsnLeuAspGlu |
| 180185190 |
| GlyMetLeuLysThrLeuGluAspLeuAspLysHisIlePheThrVal |
| 195200205 |
| AsnTyrSerAlaGluLeuAspMetMetTyrProGlyIleLeuLysLys |
| 210215220 |
| LeuAsnGlySerLeuPheProLysValValSerLysPhePheAspAsn |
| 225230235240 |
| LeuThrLysAsnValLeuGluMetArgLysGlyThrProSerTyrGln |
| 245250255 |
| LysAspMetIleAspLeuIleGlnGluLeuArgGluLysLysThrLeu |
| 260265270 |
| GluLeuSerArgLysHisGluAsnGluAspValLysAlaLeuGluLeu |
| 275280285 |
| ThrAspGlyValIleSerAlaGlnMetPheIlePheTyrMetAlaGly |
| 290295300 |
| TyrGluThrSerAlaThrThrMetThrTyrLeuPheTyrGluLeuAla |
| 305310315320 |
| LysAsnProAspIleGlnAspLysLeuIleAlaGluIleAspGluVal |
| 325330335 |
| LeuSerArgHisAspGlyAsnIleThrTyrGluCysLeuSerGluMet |
| 340345350 |
| ThrTyrLeuSerLysValPheAspGluThrLeuArgLysTyrProVal |
| 355360365 |
| AlaAspPheThrGlnArgAsnAlaLysThrAspTyrValPheProGly |
| 370375380 |
| ThrAspIleThrIleLysLysGlyGlnThrIleIleValSerThrTrp |
| 385390395400 |
| GlyIleGlnAsnAspProLysTyrTyrProAsnProGluLysPheAsp |
| 405410415 |
| ProGluArgPheAsnProGluAsnValLysAspArgHisProCysAla |
| 420425430 |
| TyrLeuProPheSerAlaGlyProArgAsnCysLeuGlyMetArgPhe |
| 435440445 |
| AlaLysTrpGlnSerGluValCysIleMetLysValLeuSerLysTyr |
| 450455460 |
| ArgValGluProSerMetLysSerSerGlyProPheLysPheAspPro |
| 465470475480 |
| MetArgLeuPheAlaLeuProLysGlyGlyIleTyrValAsnLeuVal |
| 485490495 |
| ArgArg |
| (2) INFORMATION FOR SEQ ID NO:25: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 504 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Rat |
| (B) STRAIN: |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: |
| MetAspLeuLeuSerAlaLeuThrLeuGluThrTrpValLeuLeuAla |
| 151015 |
| ValValLeuValLeuLeuTyrGlyPheGlyThrArgThrHisGlyLeu |
| 202530 |
| PheLysLysGlnGlyIleProGlyProLysProLeuProPhePheGly |
| 354045 |
| ThrValLeuAsnTyrTyrMetGlyLeuTrpLysPheAspValGluCys |
| 505560 |
| HisLysLysTyrGlyLysIleTrpGlyLeuPheAspGlyGlnMetPro |
| 65707580 |
| LeuPheAlaIleThrAspThrGluMetIleLysAsnValLeuValLys |
| 859095 |
| GluCysPheSerValPheThrAsnArgArgAspPheGlyProValGly |
| 100105110 |
| IleMetGlyLysAlaValSerValAlaLysAspGluGluTrpLysArg |
| 115120125 |
| TyrArgAlaLeuLeuSerProThrPheThrSerGlyArgLeuLysGlu |
| 130135140 |
| MetPheProIleIleGluGlnTyrGlyAspIleLeuValLysTyrLeu |
| 145150155160 |
| LysGlnGluAlaGluThrGlyLysProValThrMetLysLysValPhe |
| 165170175 |
| GlyAlaTyrSerMetAspValIleThrSerThrSerPheGlyValAsn |
| 180185190 |
| ValAspSerLeuAsnAsnProLysAspProPheValGluLysThrLys |
| 195200205 |
| LysLeuLeuArgPheAspPhePheAspProLeuPheLeuSerValVal |
| 210215220 |
| LeuPheProPheLeuThrProIleTyrGluMetLeuAsnIleCysMet |
| 225230235240 |
| PheProLysAspSerIleGluPhePheLysLysPheValTyrArgMet |
| 245250255 |
| LysGluThrArgLeuAspSerValGlnLysHisArgValAspPheLeu |
| 260265270 |
| GlnLeuMetMetAsnAlaHisAsnAspSerLysAspLysGluSerHis |
| 275280285 |
| ThrAlaLeuSerAspMetGluIleThrAlaGlnSerIleIlePheIle |
| 290295300 |
| PheAlaGlyTyrGluProThrSerSerThrLeuSerPheValLeuHis |
| 305310315320 |
| SerLeuAlaThrHisProAspThrGlnLysLysLeuGlnGluGluIle |
| 325330335 |
| AspArgAlaLeuProAsnLysAlaProProThrTyrAspThrValMet |
| 340345350 |
| GluMetGluTyrLeuAspMetValLeuAsnGluThrLeuArgLeuTyr |
| 355360365 |
| ProIleGlyAsnArgLeuGluArgValCysLysLysAspValGluIle |
| 370375380 |
| AsnGlyValPheMetProLysGlySerValValMetIleProSerTyr |
| 385390395400 |
| AlaLeuHisArgAspProGlnHisTrpProGluProGluGluPheArg |
| 405410415 |
| ProGluArgPheSerLysGluAsnLysGlySerIleAspProTyrVal |
| 420425430 |
| TyrLeuProPheGlyAsnGlyProArgAsnCysIleGlyMetArgPhe |
| 435440445 |
| AlaLeuMetAsnMetLysLeuAlaLeuThrLysValLeuGlnAsnPhe |
| 450455460 |
| SerPheGlnProCysLysGluThrGlnIleProLeuLysLeuSerArg |
| 465470475480 |
| GlnGlyLeuLeuGlnProThrLysProIleIleLeuLysValValPro |
| 485490495 |
| ArgAspGluIleIleThrGlySer |
| 500 |
| (2) INFORMATION FOR SEQ ID NO:26: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 12 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: Learn-PyR |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: |
| PheXaaXaaProXaaXaaTyrXaaProXaaArgPhe |
| 1510 |
| (2) INFORMATION FOR SEQ ID NO:27: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 1551 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA (cDNA) |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: CS |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: |
| ATGTTGTTATTACTGCTACTGATTGTGGTGACGACCCTCTACATCTTTGCCAAACTTCAT60 |
| TATACGAAATGGGAACGTTTGGGTTTCGAATCGGATAAGGCCACCATACCCCTGGGATCG120 |
| ATGGCAAAGGTGTTCCACAAGGAACGGCCATTTGGCCTGGTTATGTCCGACATATATGAC180 |
| AAATGCCACGAGAAGGTGGTGGGCATTTATTTGTTCTTCAAGCCGGCCCTACTGGTGCGC240 |
| GATGCCGAATTGGCGAGACAAATTTTGACCACGGATTTTAATAGCTTCCATGATCGTGGC300 |
| CTCTATGTGGATGAGAAAAATGATCCAATGTCGGCGAATCTTTTCGTGATGGAGGGTCAA360 |
| TCATGGCGTACGCTGAGAATGAAATTGGCCCCCTCGTTTTCGTCGGGTAAACTCAAGGGG420 |
| ATGTTCGAAACGGTCGATGATGTGGCGGATAAATTAATAAATCACTTGAATGAGCGCTTG480 |
| AAGGATGGCCAGACGCATGTTTTGGAAATCAAGAGTATTTTGACCACCTATGCTGTCGAC540 |
| ATCATTGGTTCGGTGATATTCGGCCTGGAAATCGATAGTTTCACCCATCCGGACAATGAA600 |
| TTTCGTGTCTTGAGTGATCGTCTATTTAACCCAAAGAAGTCGACAATGTTGGAGAGATTT660 |
| CGCAATTTATCAACCTTTATGTGTCCACCACTTGCCAAACTCTTGTCGCGCCTTGGTGCC720 |
| AAGGATCCGATAACATATCGCCTGCGCGACATCGTGAAACGGACGATAGAATTTCGCGAA780 |
| GAAAAGGGCGTTGTACGCAAAGATCTTCTCCAGCTATTTATACAACTCAGAAATACTGGT840 |
| AAAATTTCCGATGACAACGACAAGCTATGGCATGACGTTGAGTCGACGGCGGAAAATCTC900 |
| AAAGCCATGTCCATCGATATGATTGCCTCCAATTCATTCTTATTCTATATTGCCGGATCG960 |
| GAAACAACGGCGGCCACAACATCATTTACCATCTATGAATTGGCCATGTATCCGGAAATT1020 |
| CTGAAGAAGGCCCAAAGCGAGGTGGATGAGTGTCTGCAAAGGCACGGTCTCAAGCCGCAG1080 |
| GGACGGCTGACCTATGAGGCCATACAGGATATGAAATATTTGGATTTGTGTGTTATGGAA1140 |
| ACCACCCGCAAATACCCTGGCCTGCCGTTTTTGAATCGCAAATGCACTCAGGATTTCCAA1200 |
| GTACCCGACACAAAACTTACCATACCCAAGGAAACGGGAATTATCATCTCCCTCTTGGGC1260 |
| ATCCATAGAGACCCACAGTATTTCCCCCAACCCGAGGATTATAGGCCAGAACGCTTTGCC1320 |
| GATGAGAGCAAGGATTATGATCCAGCGGCATATATGCCTTTTGGAGAGGGTCCAAGGCAC1380 |
| TGTATTGCTCAACGCATGGGCGTTATGAATTCCAAGGTAGCCTTGGCCAAAATATTGGCC1440 |
| AATTTTAATATTCAACCAATGCCCCGCCAAGAAGTTGAGTTCAAATTCCATTCAGCTCCT1500 |
| GTTCTGGTACCAGTAAATGGTCTCAATGTGGGTCTATCGAAGAGGTGGTGA1551 |
| (2) INFORMATION FOR SEQ ID NO:28: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 517 amino acids |
| (B) TYPE: amino acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: protein |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: CS |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: |
| MetLeuLeuLeuLeuLeuLeuIleValValThrThrLeuTyrIlePhe |
| 151015 |
| AlaLysLeuHisTyrThrLysTrpGluArgLeuGlyPheGluSerAsp |
| 202530 |
| LysAlaThrIleProLeuGlySerMetAlaLysValPheHisLysGlu |
| 354045 |
| ArgProPheGlyLeuValMetSerAspIleTyrAspLysCysHisGlu |
| 505560 |
| LysValValGlyIleTyrLeuPhePheLysProAlaLeuLeuValArg |
| 65707580 |
| AspAlaGluLeuAlaArgGlnIleLeuThrThrAspPheAsnSerPhe |
| 859095 |
| HisAspArgGlyLeuTyrValAspGluLysAsnAspProMetSerAla |
| 100105110 |
| AsnLeuPheValMetGluGlyGlnSerTrpArgThrLeuArgMetLys |
| 115120125 |
| LeuAlaProSerPheSerSerGlyLysLeuLysGlyMetPheGluThr |
| 130135140 |
| ValAspAspValAlaAspLysLeuIleAsnHisLeuAsnGluArgLeu |
| 145150155160 |
| LysAspGlyGlnThrHisValLeuGluIleLysSerIleLeuThrThr |
| 165170175 |
| TyrAlaValAspIleIleGlySerValIlePheGlyLeuGluIleAsp |
| 180185190 |
| SerPheThrHisProAspAsnGluPheArgValLeuSerAspArgLeu |
| 195200205 |
| PheAsnProLysLysSerThrMetLeuGluArgPheArgAsnLeuSer |
| 210215220 |
| ThrPheMetCysProProLeuAlaLysLeuLeuSerArgLeuGlyAla |
| 225230235240 |
| LysAspProIleThrTyrArgLeuArgAspIleValLysArgThrIle |
| 245250255 |
| GluPheArgGluGluLysGlyValValArgLysAspLeuLeuGlnLeu |
| 260265270 |
| PheIleGlnLeuArgAsnThrGlyLysIleSerAspAspAsnAspLys |
| 275280285 |
| LeuTrpHisAspValGluSerThrAlaGluAsnLeuLysAlaMetSer |
| 290295300 |
| IleAspMetIleAlaSerAsnSerPheLeuPheTyrIleAlaGlySer |
| 305310315320 |
| GluThrThrAlaAlaThrThrSerPheThrIleTyrGluLeuAlaMet |
| 325330335 |
| TyrProGluIleLeuLysLysAlaGlnSerGluValAspGluCysLeu |
| 340345350 |
| GlnArgHisGlyLeuLysProGlnGlyArgLeuThrTyrGluAlaIle |
| 355360365 |
| GlnAspMetLysTyrLeuAspLeuCysValMetGluThrThrArgLys |
| 370375380 |
| TyrProGlyLeuProPheLeuAsnArgLysCysThrGlnAspPheGln |
| 385390395400 |
| ValProAspThrLysLeuThrIleProLysGluThrGlyIleIleIle |
| 405410415 |
| SerLeuLeuGlyIleHisThrHisProGlnTyrPheProGlnProGlu |
| 420425430 |
| AspTyrArgProGluArgPheAlaAspGluSerLysAspTyrAspPro |
| 435440445 |
| AlaAlaTyrMetProPheGlyGluGlyProArgHisCysIleAlaGln |
| 450455460 |
| ArgMetGlyValMetAsnSerLysValAlaLeuAlaLysIleLeuAla |
| 465470475480 |
| AsnPheAsnIleGlnProMetProArgGlnGluValGluPheLysPhe |
| 485490495 |
| HisSerAlaProValLeuValProValAsnGlyLeuAsnValGlyLeu |
| 500505510 |
| SerLysArgTrpXaa |
| 515 |
| (2) INFORMATION FOR SEQ ID NO:29: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 2085 base pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (ii) MOLECULE TYPE: DNA (genomic) |
| (iii) HYPOTHETICAL: NO |
| (iv) ANTI-SENSE: NO |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: Musca domestica |
| (B) STRAIN: Learn-PyR |
| (D) DEVELOPMENTAL STAGE: Adult |
| (viii) POSITION IN GENOME: |
| (A) CHROMOSOME/SEGMENT: Chromosome 1 |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: |
| GACAGGGAGAAGGAAATGATAAGAAATGTGCAAAGTTTTACATGCATTCGAATCATTCTG60 |
| TTTCACAAAATGACCGGCAACTATTCAGTTGTTAATGTAACACGTACCCGATTAGATCGG120 |
| AAATATTTTGTAACGTTAGTGAATAGCTAGGAGAAAAATTGCAAAACTAAAATGTTGTTA180 |
| TTACTGCTACTGATTGTGGTGACGACCCTCTACATCTTTGCCAAACTTCATTATACGAAA240 |
| TGGGAACGTTTGGGTTTCGAATCGGATAAGGCCACCATACCCCTGGGATCGATGGCAAAG300 |
| GTGTTCCACAAGGAACGGCCATTTGGCCTGGTTATGTCAGACATATACGACAAATGCCAC360 |
| GAGAAGGTGGTGGGCATTTATTTGTTCTTCAAGCCGGCCCTACTGGTGCGCGATGCCGAA420 |
| TTGGCGAGACAAATTTTGACCACGGATTTTAATAGCTTCCATGATCGTGGCCTCTATGTG480 |
| GATGAGAAAAATGATCCAATGTCGGCGAATCTTTTCGTGATGGAGGGTCAATCATGGCGT540 |
| ACGCTGAGAATGAAATTGGCCCCCTCGTTTTCGTCGGGTAAACTCAAGGGGATGTTCGAA600 |
| ACGGTCGATGATGTGGCGGATAAATTAATAAATCACTTGAATGAGTGCTTGAAGGATGGC660 |
| CAGACGCATGTTTTGGAAATCAAGAGTATTTTGACCACGTAAGTACTCATCGTTGAGAGA720 |
| ATTGTAAGAAGTTTTGAATTTTACTTTTAATAAAATGTTCTTCTTCCCCCAGCTATGCTG780 |
| TCGACATCATTGGTTCGGTGATATTCGGCCTGGAAATCGATAGTTTCACCCATCCGGACA840 |
| ATGAATTTCGTGTCTTGAGTGATCGTCTATTTAACCCAAAGAAGTCGACAATGTTGGAGA900 |
| GATTTCGCAATTTATCAACCTTTATGTGTCTACCACTTGCCAAACTCTTGTCGCGCCTTG960 |
| GTGCCAAGGATCCGATAACATATCGCCTGCGCGACATCGTGAAACGGACGATAGAATTTC1020 |
| GCGAAGAAAAGGGCGTTGTACGCAAAGATCTTCTCCAGCTATTTATACAACTCAGAAATA1080 |
| CTGGTAAAATTTCCGATGACAACGACAAGCTATGGCATGACGTTGAGTCGACGGCGGAAA1140 |
| ATCTCAAAGCCATGTCCATCGATATGATTGCCTCCAATTCATTCTTATTCTATATTGCCG1200 |
| GATCGGAAACAACGGCGGCCACAACATCATCTACCATCTATGAATTGGCCATGTATCCGG1260 |
| AAATTCTGAAGAAGGCCCAAAGCGAGGTGGATGAGTGTCTGCAAAGGCACGGTCCCAAGC1320 |
| CGCAGGGACGGCTGACCTATGAGGCCATACAGGATATGAAATATTTGGATTTGTGTGTTA1380 |
| TGGGTAAGAGGGGAAATTTTGAAATTGTTTTTTTTTTTTATTTTCTAATTATTGCATGTT1440 |
| TTTGTTGTAGAAACCACCCGCAAATACCCTGGCCTGCCGTTTTTGAATCGCAAATGCACT1500 |
| CAGGATTTCCAAGTACCCGACACAAAACTTACCATACCCAAGGAAACGGGAATTATCATC1560 |
| TCCCTCTTGGGCATCCATAGAGACCCACAGTATTTCTCCCAACCCGAGGATTATAGGCCA1620 |
| GAACGCTTTGCCGATGAGAGCAAGGATTATGATCCAGCGGCATATATGCCTTTTGGAGAG1680 |
| GGTCCAAGGCACTGTATTGGTGAGATGTTGAAAGGGGAGCTTCATTAAATTCTGAATATT1740 |
| AATTTTGTATTTTTTTTCCACACCGCTCAACGCATGGGCGTTATGAATTCCAAGGTAGCC1800 |
| TTGGCCAAAATATTGGCCAATTTTAATATTCAACCAATGCCCCGCCAAGAAGTTGAGTTC1860 |
| AAATTCCATTCAGCTCCTGTTCTGGTACCAGTAAATGGTCTCAATGTGGGTCTATCGAAG1920 |
| AGGTGGTGAAGAGCAAGTGGTTAAGTGAATTGAGGAGTGCTTTTTCGAGATATATGTTGG1980 |
| TGATTAGGGTTATAACGATTATTTAAGAACCAGTATTTAAGCTTTAATTTTTTATTCAAA2040 |
| ATTTTTGAAATATTGAAATTAAAATAAACATATGTAAATAAAATT2085 |
| __________________________________________________________________________ |
Tomita, Takashi, Scott, Jeffrey G.
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